For Review Only - digital.csic.esdigital.csic.es/bitstream/10261/47373/3/Ketoconazole_Inhibits.pdf · 56 azol-derivative antifungal agent with a broad spectrum of activity against

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HONTORIA ET AL.--- EFFECT OF KETOCONAZOLE ON ICHTHYOPHONUS 1

2

Ketoconazole Inhibits the Growth and Development of Ichthyophonus sp. 3

(Mesomycetozoa) in Vitro 4

5

FRANCISCO HONTORIA, Mª ANGELES GONZÁLEZ, ARIADNA SITJÀ-BOBADILLA, 6

OSWALDO PALENZUELA, PILAR ALVAREZ-PELLITERO 7

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Instituto de Acuicultura Torre de la Sal (CSIC), 12595 Ribera de Cabanes, Castellón, Spain 9

10

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12

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Corresponding author: P. Alvarez-Pellitero, Instituto de Acuicultura Torre de la Sal (CSIC), 18

12595 Ribera de Cabanes, Castellón, Spain---Telephone number:+34-964319500, FAX 19

number: +34-964319509; e.mail:[email protected] 20

Page 1 of 26 Journal of Eukaryotic Microbiology

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ABSTRACT. 21

Key words. Teleostei, treatments. 22

The in vitro effect of the azol-derivative antifungal Ketoconazole (KZ) on the morphology, 23

growth and development of Ichthyophonus sp, was studied. KZ was delivered to culture 24

medium using liposomes (L) or a lipid emulsion (E). Five different KZ doses (5, 50, 100, 200 25

and 400 µg/ml) were assayed with both L and E formulations. Controls consisted of MEM-10 26

alone (C-MEM) or containing the amounts of L or E equivalent to those used in the KZ100 27

and KZ400 treatments (100L, 400L, 100E and 400E, respectively). Clear morphological 28

alterations were observed by light and electron microscopy in the MEM-cultured organisms 29

receiving KZ formulations, specially with KZ400L preparations, both at 7 and 14 days post-30

inoculation. KZ also inhibited Ichthyophonus growth in MEM and its effect was statistically 31

significant with respect to the corresponding controls. KZ also had an inhibitory effect on 32

subsequent Ichthyophonus germination in EFSA medium, which was more evident for L 33

formulations when the organism was treated for 7 days and for E formulations in the 14 days 34

treatment. The obtained results endorse the potential use of KZ for the treatment for 35

ichthyophonosis and the interest of confirming it using in vivo assays. 36

Page 2 of 26Journal of Eukaryotic Microbiology

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Ichthyophonus is an obligate parasite with a wide host spectrum, including some 37

freshwater or anadromous fish and numerous marine fish (McVicar, 1998). The taxonomic 38

position and identity of Ichthyophonus have been very controversial since its first description 39

from Salmo trutta by Hofer (1893). Firstly considered as a member of Protozoa (Caullery and 40

Mesnil 1905), the organism was ascribed to Fungi by Plehn and Mulsow (1911), but this 41

ascription was further questioned (reviewed in McVicar 1998). Molecular studies have 42

demonstrated that Ichthyophonus and other related microbes constitute a phylogenetic group 43

in the boundaries of the animal-fungal divergence, which has been referred to as the DRIP 44

clade (Ragan et al. 1996; Spanggaard et al. 1996). Ichthyophonus was later ascribed to the 45

class Mesomycetozoa (Mendoza et al. 2002) and this class was included within Opisthokonta 46

and not in Fungi in the new classification of protists (Adl et al. 2005). 47

Information on treatment and control methods for ichthyophonosis is very scarce. 48

Some treatments suggested for the first infection steps, such as fenoxetol and 49

paraclorofenoxetol or antibiotics (Van Duijn 1956) proved not to be very effective. Therefore, 50

prophylactic measures such as food pasteurisation (McVicar 1982) or disinfection 51

(Hersheberger, Pacheco and Gregg 2008) were suggested. In vitro studies demonstrated the 52

inhibitory activity of the antifungal ketoconazole (KZ) on Ichthyophonus growth (Chauvier 53

and Mortier-Gabet 1982). The preliminary in vitro and in vivo studies of Franco-Sierra (1994) 54

also pointed to a promising effect of KZ, especially when vehiculated in liposomes. KZ is an 55

azol-derivative antifungal agent with a broad spectrum of activity against both superficial and 56

systemic micosis. Its antifungal action is related to the inhibition of cytochrome P-450-57

dependent demethylation of lanosterol in the biosynthetic pathway of ergosterol in fungi 58

(Vanden Bossche et al. 1988). However, it also affects a number of mammalian cytochromes, 59

and thus can cause liver damage through inhibition of NADH oxidase and thus mitochondrial 60

activity (Rodríguez and Acosta 1996). 61


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Liposomes, as vesicles formed by polar lipids in bilayer arrangements enclosing 62

aqueous compartments, can be manipulated to encapsulate different substances that are 63

isolated from the surrounding medium. Liposomes were first proposed and tested as a drug 64

delivery system more than 30 years ago. Since then, the design of constructs for use in the 65

treatment and prevention of disease has substantially improved (Gregoriadis 1995; Torchilin 66

2005) and a reduction of toxicity of several drugs when encapsulated in liposomes has been 67

reported (Mehta 1996). Consequently, the use of liposomes as delivering agent for KZ can 68

provide different advantages related to the protection against the drug toxicity, and to the 69

improvement of the treatment efficiency due to the facility of the liposomes to reach the cells 70

and the intracellular pathogens. 71

We have reported the finding of an Ichthyophonus sp. from grey mullets and other 72

marine fish from the Mediterranean area, and provided data on the infection in different 73

mariculture systems (Franco-Sierra, Sitjà-Bobadilla and Alvarez-Pellitero 1997). A detailed 74

morphological study, including ultrastructural data has also been presented (Franco-Sierra and 75

Alvarez-Pellitero 1999). The available data on the KZ properties and the advantages of 76

liposome delivering prompted us to afford in depth studies on the effect of KZ on this 77

Mediterranean icthyophonosis, using in vitro and in vivo methods. In the present work, the 78

effect of the drug on the growth of Ichthyophonus cultured in vitro is evaluated. In addition, 79

the effect of the treatment on the morphology of cultured stages is studied at light and 80

transmission electron microscopes. 81

82

MATERIAL AND METHODS 83

84 Preparation of liposomes and ketoconazole (KZ)-formulations. Five different KZ 85

doses (5, 50, 100, 200 and 400 µg/ml) were prepared in MEM-10 (see below). Two different 86


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KZ formulations were used to deliver the drug in the culture medium, liposomes (L) or an 87

equivalent lipid emulsion (E). Thus, ten KZ formulations (KZ5L, KZ50L, KZ100L, KZ200L, 88

KZ400L, KZ5E, KZ50E, KZ100E, KZ200E and KZ400 E) were assayed. Controls consisted 89

of MEM-10 alone (C-MEM) or containing the amounts of L or E equivalent to those used in 90

the KZ100 and KZ400 treatments (100L, 400L, 100E and 400E, respectively) (Table 1). 91

Liposomes were prepared with phosphatidylcholine extracted from egg yolk (EPC) 92

purchased from Avanti Polar Lipids Inc. (Alabaster, Alabama USA). Palmiticic (16:0, 34% of 93

total fatty acids) and oleicic (18:1, 31%) acids are the main fatty acids of EPC. It also contains 94

18% of linoleic acid (18:2). Cholesterol (CHO), purchased from Sigma-Aldrich Química S.A. 95

(Alcobendas, Madrid, Spain), was included as a membrane stabilizer, and stearylamine (ST), 96

a polar derivative from the stearic acid also from Sigma-Aldrich Química S.A., as charge to 97

prevent the aggregation. In the case of the treatment formulations, 20% w/w of ketoconazole 98

(Acofarma, Barcelona, Spain), was also incorporated to the liposome composition. The 99

liposomes under the form of multilamellar vesicles (MLV) were prepared by the method 100

proposed by Bangham et al. (1965), but using MEM-10 as the aqueous phase. The lipid 101

mixture was dried under nitrogen flux in a thin layer on the bottom of a flask and rehydrated 102

with the aqueous phase during one hour by vortexing frequently until an homogenous 103

suspension was achieved. The final composition of the lipid mixture for the mother 104

preparation used in the liposomes for treatment doses was: EPC: KZ: CHO: ST (56:20:19:5 105

w/w), and in the control liposomes without KZ: EPC: CHO: ST (76:19:5 w/w). This mixture 106

was then rehydrated with MEM-10 and used in the necessary amounts to reach the 107

experimental doses in each culture well (see below). The lipid emulsions were prepared 108

emulsifying the triacylglyceride triolein (TON) with Tween 80 (polysorbate 80), both 109

acquired from Sigma-Aldrich Química S.A., in MEM-10, using an IKA Ultra-Turrax tissue 110

disruptor (IKA Laborteknik, Staufen, Germany) at high lipidic concentration in order to 111


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improve the stability. The final composition of this mother emulsion was: MEM: TON: 112

Tween80 (89.6:7.1:3.3 v/v). A similar mother emulsion was prepared for the treatment 113

emulsions. In this case, KZ was added to achieve a final concentration of 4 mg/ml. Both 114

emulsions were then diluted with MEM in each culture well to achieve the appropriate 115

experimental doses. The different concentrations of the liposomes or emulsion components in 116

each treatment well are also shown in Table 1. 117

Culture media. The organism was cultured in Eagle’s minimum essential medium 118

(Sigma M5775) (MEM) at pH 7.2 supplemented with HEPES (Gibco) (20 mM), 10 % foetal 119

bovine serum (FBS) (Sigma F4010) (MEM-10) and gentamycin (50 µg.ml-1). Earle’s fish 120

saline agar (EFSA), prepared as in Rand and Cone (1990) and supplemented with 10 % FBS, 121

was used to evaluate germination. 122

Ichthyophonus source. The Ichthyophonus used in this study was aseptically taken 123

and isolated from organs of naturally infected mullets suffering an epizootic in August 1990 124

(Franco-Sierra et al., 1997). Since the original isolation, it has been kept in vitro through 125

serial passages in 100 ml bottles containing MEM-10 (see above) and under the conditions 126

selected in a previous study (Franco-Sierra and Alvarez-Pellitero, 1999) (pH 7.2 and 14 ºC). 127

Ichthyophonus from routine cultures was replicated in the same medium and conditions in T-128

25 flasks to obtain the amount necessary for the assays. 129

Evaluation of growth. Tests were performed in six well plates containing 3 ml of 130

MEM-10 per well with the appropriate amounts of the corresponding mother preparations to 131

get the necessary concentrations of each treatment, or MEM-10 alone (C-MEM). The 132

resulting concentrations of each component of E or L in the corresponding control and KZ 133

formulations are detailed in Table 1. Each well was seeded with 100 µl of Ichthyophonus 134

culture containing 1.58 × 106 spores/ml. Three replicates per treatment were incubated at 14ºC 135

during 7 days and the other three replicates during 14 days. The development of the organism 136


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was followed using an inverted microscope (×100, Nikon Diaphot-TMD). At the end of each 137

incubation period, the Ichtyophonus stages present in each well were collected and the 138

number of viable stages was counted in a Neubauer chamber using the eosin-exclusion 139

method. Part of this material was used to further evaluate germination (see below). Samples 140

were also taken for fresh examination by light microscope (LM) using a Leitz Dialux 22 141

microscope. For transmission electron microscope (TEM studies), samples from 14 days 142

cultures corresponding to each treatment were pelleted at 700 g for 5 minutes at 4°C. Pellets 143

were fixed in 2.5 % v/v glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2, 4 °C) for 12 h., 144

postfixed in 0.1% (w/v) cacodylic OsO4 for 2 h, dehydrated through a graded ethanol series 145

and embedded in Spurr’ resin (Spurr 1969). Ultrathin sections were double stained with 146

aqueous uranyl acetate and lead citrate (Reynolds 1963). Some grids were stained by the OTO 147

technique for lipids (Seligman et al. 1966)). The sections were studied in a Philips CM 200 or 148

a Hitachi AD 600 transmission electron microscopes, operating between 60 and 75 kV. 149

Evaluation of further germination. To evaluate the further development of 150

Ichthyophonus after 7 or 14 days of treatment, an equal number of Ichthyophonus viable 151

stages from each treatment and each incubation period were seeded by triplicate on Petri 152

dishes containing EFSA and incubated at 14 ºC. The volume to be seeded in each case was 153

deduced from the counting data indicated above and was calculated to obtain 7.75 × 104 154

stages per plate. Growth was evaluated as the number of colony forming units (CFU)/ml at 14 155

days post-seeding. The development of the organism was followed using an inverted 156

microscope as above. 157

Statistical analysis. The number of viable spores or organism-units per ml grown in MEM 158

(mean of the three replicates) in each treatment after 7 and 14 days was compared using two 159

separate one-way ANOVA tests with the Brown-Forsythe transformation in search of 160

differences among treatments followed by the Games-Howell’s test for multiple mean 161


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comparisons when appropriate. Both tests are robust in cases of variance heteroscedasticity, 162

which have been the case in our study due to the high dispersion that these traits show. 163

Differences in the number of CFU/ml observed in EFSA (mean of the three replicates) after 164

incubation periods of 7 and 14 days were also analysed with the same tests. 165

166

RESULTS 167

168

Morphological studies. The effect of the KZ treatment on Ichthyophonus structures 169

was evidenced at both LM and TEM. At LM, spores in the different stages of growth as well 170

as the typical development (division, budding) were seen in C-MEM (Fig. 1). No appreciable 171

changes were induced by the lowest dose (KZ5), but both the spore morphology and the 172

development were altered starting with the KZ50 dose and damage clearly increased with 173

higher doses. Changes consisted mainly of a decrease in the number of dividing and budding 174

spores and an alteration of the spore structure, i.e. retraction and densification of the 175

cytoplasmic material. Such alterations were evident with KZ100 dose and more pronounced 176

with KZ400 dose, being the effect more intense with L (Fig. 2) than with E preparations (Fig. 177

3). The addition of 100L and 400L alone also induced some alterations in the spores (Fig.4), 178

not or scarcely observed with 100E and 400E (Fig. 5). The noxious effect of KZ was already 179

observed at 7 days post-inoculation (p.i) and was more pronounced at 14 days p.i. 180

To more accurately evaluate the effect of KZ on Ichthyophonus stages, a TEM study 181

was carried out with the material from the different treatments collected at 14 days p.i. In 182

addition, the ability of the organism to germinate was evaluated after seeding in EFSA. In the 183

TEM study, spores cultured in C-MEM had the typical structure (Fig. 6, 7), with abundant 184

nuclei, some endospores, and a well defined wall. The cytoplasm was densely packed with 185

ribosomes and abundant glycogen. Mitochondria, endoplasmic reticulum some vacuoles and 186


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several lipid droplets (as confirmed with OTO staining) were also observed. The addition of 187

KZ produced appreciable effect on spores from the KZ100 dose on. With KZ100E, the 188

number of nuclei was substantially reduced indicating a decrease in cell activity. This effect 189

was more pronounced with KZ400E. In some spores the cytoplasm became amorphous (Fig. 190

8) and a destruction of the cells contents occurred, though abundant lipidic vesicles were still 191

evidenced by OTO staining (Fig. 9). The addition of KZ100L and KZ400 L induced even 192

more evident alterations of the spores, consisting of condensation of the cytoplasmic contents 193

in some thick-walled spores, or destruction of the cytoplasmic contents and wall (Fig.10). As 194

observed by LM, the addition of 100L and 400L produced also alterations in some spores, 195

mainly condensation of the cytoplasm contents (Fig. 11). Spores maintained in 100E and 196

400E, did not show such alterations but abundant lipidic droplets (Fig. 12). 197

Differences in the germination of Ichthyophonus in EFSA were observed after 14 days 198

of KZ treatments with respect to controls. Ichthyophonus previously cultured in C-MEM 199

showed the typical germination pattern, with formation of long septate hyphae, which, after 200

branching, developed spores in the bulbous tips (Fig. 13). In contrast, many spores previously 201

cultured in KZ100E were not able to germinate (Fig. 14) and no germination was observed in 202

KZ100L (Fig. 15), KZ400E (Fig. 16) and KZ400L (Fig. 17) samples. 203

Evaluation of growth in MEM-10 and further germination in EFSA. At 7 days p.i., 204

an stimulating effect of lipid addition on Ichthyophonus growth was observed in both control 205

L and E treatments with respect to C-MEM, though the difference was statistically significant 206

only for 400L and 400E. Therefore, the effect of KZ treatment must be compared with the 207

corresponding controls with added liposomes or lipids. Thus, the addition of KZ significantly 208

inhibited the growth with respect to L and E controls even at the lowest dose (5KZ) (Fig. 209

18A). At 14 days of treatment, the effect of the of L or E alone on the growth was slight, and 210

no statistically significant differences were detected with respect to C-MEM. An inhibitory 211


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effect of KZ treatment was also detected, but differences were statistically significant only for 212

the highest doses of E treatments, KZ200E and KZ400E (Fig. 18B). A quite good dose/effect 213

correlation was generally observed. 214

The further viability of spores was evaluated in EFSA. The germination of Ichthyophonus 215

that have been kept for 7 days in KZ100E, KZ200E and KZ400E was significantly lower than 216

in the corresponding E controls and in the C-MEM. At this time, most L treatments had 217

significantly lower values than those of C-MEM (Fig. 19A). The effect of KZE was more 218

evident for Ichthyophonus that have been kept for 14 days in the corresponding treatments, as 219

the number of CFU was significantly lower than in E controls for all the doses assayed, and 220

also with respect to C-MEM for the highest doses (Fig. 19B). No significant differences were 221

found at this time between treated and untreated L groups. However, the CFU number was 222

significantly lower for the three highest KZ doses (100L, 200L and 400L) with respect to C-223

MEM (Fig. 19B). 224

225

DISCUSSION 226

The obtained results demonstrated that KZ inhibited the growth and development of 227

Ichthyophonus cultivated in vitro. The noxious effect was higher when KZ was delivered in 228

liposomes than when incorporated in the lipid emulsion. Both the growth kinetics and 229

morphological studies evidenced that KZ has a clear effect on this organism and that this 230

effect is influenced by the delivery system. To check the possible advantages of liposomal (L) 231

delivering, KZ had to be incorporated to the culture medium using a lipid emulsion (E) 232

vehicle, as the drug is not hydro soluble. Multilamellar vesicle (MLV) liposomes can 233

encapsulate lipophyllic materials as KZ more readily than unilamellar liposomes. The non 234

polar core of the multiple MLV membranes offers more encapsulation space for KZ. The 235


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positive charge afforded by the sterylamine grants a repulsive force between the different 236

membranes that prevents liposome aggregation. 237

The addition of both L and E alone stimulated the growth and development of 238

Ichthyophonus in MEM at 7 days p.i., as the number of viable units was higher, both in L and 239

E controls than in C-MEM. In contrast, this effect was negligible at 14 days p.i. Lipid 240

emulsion addition did not induce important morphological changes, apart from the increasing 241

of lipidic inclusions in the cytoplasm, but the presence of L induced some changes in the 242

organism. It seems that this additional lipid supply favours the growth of Ichthyophonus, at 243

least initially. However, such effect was not so clear at longer term, mainly for L preparations, 244

as germination was lower than in C-MEM both after 7 and 14 days in such condition. For E 245

preparations, germination was better for 100E than for 400E. Such results in control L and E 246

formulations seem to indicate that an excess of lipids might be counterproductive and 247

overpass the clear lipasic activity that was demonstrated in previous metabolic studies of 248

Ichthyophonus (Franco-Sierra 1994). The effect of L on Ichthyophonus growth after 14 days 249

of treatment and in germination at both times might be due to some of the components used in 250

its preparation. The cholesterol concentration in C400L is 0.376 mg/ml, which could be 251

excessively high for a good performance of the organism, as in the case of 252

phosphatidylcholine (1.92 mg/ml). However, stearylamine probably has no effect at the used 253

concentrations, and has been reported as a liposome component improving some effects as the 254

DNA transfection to eukaryotic cells (Wang, Jin and Lin, 1996). 255

Both KZL and KZE formulations produced a decreasing in the Ichthyophonus 256

growth with respect to the corresponding L and E controls, though this effect was statistically 257

significant only for the highest doses. Such effect was confirmed by the results of spore 258

germination, mainly in the case of KZE formulations. The antifungal action of KZ and other 259

imidazol derivatives is related to the inhibition of the biosynthesis of ergosterol, a major sterol 260


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in fungi. As fungal sterols are structurally distinct from their mammalian counterparts, their 261

synthesis has been a major target for antifungal drug development (Oehlschlaeger and 262

Czyzewska, 1992). Like cholesterol in higher organisms, ergosterol is a component of 263

membranes and other cellular organelles in fungi and other microorganisms. However, it has 264

a different biosynthetic pathway due the presence of a ∆-24(25)-sterol methyltransferase 265

enzyme catalysing the incorporation of a methyl group in the side chain of the steroid nucleus, 266

which is absent in the mammalian cells (Barrett-Bee and Ryder, 1992) . The proposed 267

mechanism of action is the inhibition of cytochrome P-450-dependent demethylation of 268

lanosterol to ergosterol by the 14-α-demethylase system. Inhibition of ergosterol synthesis 269

may result in direct damage to the fungus. Indirect injury can also be produced by 270

accumulation of 14-α-methyl sterols. Other important enzymes may also be affected (Feldman 271

1986; Vanden Bossche et al. 1988). Although the mechanisms involved in the observed action 272

of KZ on the Ichthyophonus growth and development are unknown, the participation of the 273

inhibition of cytochrome P-450 enzymes as described in fungi cannot be disregarded. As a 274

matter of fact, an effect of imidazoles on cytochorome P450-related biotransformation 275

processes has been described in other systems and even in mammals (Fink-Gremmels 2008), 276

resulting in the inhibition of the mitochondrial functions (Feldman 1986; Rodriguez and 277

Acosta 1996). Ichthyophonus was initially classified within Fungi, but currently is considered 278

to belong to Mesomycetozoa. Scarce data are available on the metabolic pathways of 279

Ichthyophonus. Franco-Sierra (1994) found slight phosphatase and clear lipase activities in 280

this organism, but the lipidic composition of the membranes, and thus the presence of 281

ergosterol is unknown. 282

The morphologic alterations observed in Ichtyophonus treated in KZ-containing 283

MEM-formulations consisted mainly of the loss of refringent bodies, darkening and 284

condensation of the cytoplasm and alterations of the membrane, and were related with the 285


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impairment of cellular activity. The effect on further germination in EFSA plates was 286

dramatic for KZ100L, KZ400L and KZ400E formulations, though some typical germination 287

was still observed with KZ100E treatment. The ultrastructural study, corresponding to the 288

material treated in KZ-containing MEM-formulations during 14 days, demonstrated the loss 289

of cytoplasmic integrity, which appeared completely withered in some spores, in contrast with 290

the dense cytoplasmic contents observed in control media. Glycogen granules, very abundant 291

as reserve in control organisms, were very scarce or absent in treated spores, in which, 292

furthermore, very few nuclei, organelles and ribosomes were observed. In addition, different 293

degrees of disorganization were detected in the spore wall. Ultrastructural changes induced by 294

KZ have been reported in other in vitro cultured parasites, such as Leishmania, consisting of 295

the appearance of large multivesicular bodies, increased amount of lipid inclusions, and 296

alterations in the distribution and appearance of mitochondrial cristae, accompanied by strong 297

phosphatase acid activity (Vannier-Santos et al. 1995). Another azole derivate, itraconazole, 298

provokes pellicle disruption in Toxoplasma gondi and affects the parasite division by 299

endodyogeny (Martins-Duarte, de Souza and Vommaro 2008). Different alterations have been 300

described in Leishmania amazonensis treated with sterol methenyl transferase inhibitors, 301

mainly associated with mitochondria (Rodrigues et al., 2007). In addition, alterations in 302

organelles such as nucleus, endoplasmic reticulum and Golgi complex have also been 303

described in other parasites with different drugs related or not with ergosterol byosynthesis 304

(de Carvalho et al. 2005; Martins-Duarte et al. 2006; Granthom et al. 2007). The scarcity of 305

organelles in KZ treated Ichthyophonus difficults the evaluation of damage at that level. 306

In conclusion, KZ proved to be effective in inhibiting the growth and development of 307

Ichthyophonus sp. in vitro. Both delivering systems, liposomes and lipid emulsion can be 308

successfully used to vehiculate the drug. Under the light of the obtained results, further in vivo 309

studies are needed to ascertain the potential of KZ as treatment for ichthyophonosis . 310


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ACKNOWLEDGEMENTS 311

Funding for this work was obtained from the project PTR93-0073 of the PETRI Program 312

(Spanish Government R+D National Plan). 313

314

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Figure legends 412

Fig. 1-5. Effect of KZ on the Ichthyophonus growth in MEM-10. 1. Normal growth with 413

division and budding in control (C-MEM) cultures. 2, 3. The effect of KZ is evident in 414

KZ400L (2) and KZ400E (3) treatments. 4. Spore morphology is somewhat altered in 400L. 415

5. In 400E, spores have a normal appearance and can experience budding. Scale bars = 50 416

µm. 417

Fig. 6-12. TEM images of Ichthyophonus kept for 14 days in C-MEM or in media with E and 418

L or KZ formulations. 6, 7. Morphology of spores in C-MEM showing abundant ribosomes, 419

glycogen granules and several nuclei (arrowheads). The presence of lipid inclusions is 420

demonstrated by OTO staining (7). 8, 9. The effect of KZ is observed KZ400 E treatment. 421

The wall structure is altered and the cytoplasm losses its integrity. Lipid inclusions can be 422

abundant, as demonstrated with OTO staining (9). Arrowhead points to a nuclei. 10. The 423

effect of KZ400L is dramatic. The wall is altered and the cytoplasm appears disintegrated, 424

with visible organelles and very scarce nuclei (arrowhead). 11. Spores in 400L show altered 425

morphology, with disorganization of the wall and cytoplasm. Arrowheads point to nuclei . 12. 426

Spores in 400E show a normal morphology, though lipidic inclusions are very abundant. 427

Arrowheads point to nuclei. Scale bars: Figs. 6-9, 11-12 = 1 µm; Fig. 10 = 4 µm. 428

Fig. 13-17. Germination of Ichthyophonus in EFSA medium after maintenance in C-MEM or 429

in media with KZ formulations. 13. The organism from C-MEM shows the typical 430

germination with formation of dichotomic hyphae.14-17. Germination decreases in the 431

organisms previously kept in KZ formulations. Germination is still observed for KZ100E 432

(14), but is almost absent for KZ100L (15) and specially for KZ400E (16) and KZ400L (17). 433

Scale bars = 100 µm. 434

Fig. 18. Number of Ichthyophonus viable units (mean ± SD) present in C-MEM cultures after 435

7 (A) and 14 (B) days of treatment in KZ or E and L formulations. Different letters indicate 436


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statistically significant differences (P≤0.05) after the one-way ANOVA tests and the multiple 437

comparisons of means. Solid lines indicate the control (C-MEM) mean values of the 438

triplicates and dotted lines their associated standard deviations. 439

Fig. 19. Number of Ichthyophonus colony forming units (CFU) (mean ± SD) germinated in 440

EFSA after previous treatment with the KZ or E and L formulations during 7 (A) or 14 (B) 441

days. Different letters indicate statistically significant differences (P≤0.05) after the one-way 442

ANOVA tests and the multiple comparisons of means. Solid lines indicate the control (C-443

MEM) mean values of the triplicates and dotted lines their associated standard deviations. 444


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Fig. 1-5. Effect of KZ on the Ichthyophonus growth in MEM-10. 1. Normal growth with division and budding in control (C-MEM) cultures. 2, 3. The effect of KZ is evident in KZ400L (2) and KZ400E (3) treatments. 4. Spore morphology is somewhat altered in 400L. 5. In 400E, spores have a normal

appearance and can experience budding. Scale bars = 50 µm. 88x172mm (300 x 300 DPI)


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Fig. 6-12. TEM images of Ichthyophonus kept for 14 days in C-MEM or in media with E and L or KZ formulations. 6, 7. Morphology of spores in C-MEM showing abundant ribosomes, glycogen granules and several nuclei (arrowheads). The presence of lipid inclusions is demonstrated by OTO staining (7). 8, 9. The effect of KZ is observed KZ400 E treatment. The wall structure is altered and the

cytoplasm losses its integrity. Lipid inclusions can be abundant, as demonstrated with OTO staining (9). Arrowhead points to a nuclei. 10. The effect of KZ400L is dramatic. The wall is altered and the cytoplasm appears disintegrated, with visible organelles and very scarce nuclei (arrowhead). 11.

Spores in 400L show altered morphology, with disorganization of the wall and cytoplasm. Arrowheads point to nuclei . 12. Spores in 400E show a normal morphology, though lipidic

inclusions are very abundant. Arrowheads point to nuclei. Scale bars: Figs. 6-9, 11-12 = 1 Î¼m; Fig. 10 = 4 Î¼m.

170x252mm (300 x 300 DPI)


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Figs. 13-17. Germination of Ichthyophonus in EFSA medium after maintenance in C-MEM or in media with KZ formulations. 13. The organism from C-MEM shows the typical germination with

formation of dichotomic hyphae.14-17. Germination decreases in the organisms previously kept in KZ formulations. Germination is still observed for KZ100E (14), but is almost absent for KZ100L

(15) and specially for KZ400E (16) and KZ400L (17). Scale bars = 100 µm. 142x219mm (300 x 300 DPI)


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Fig. 18. Number of Ichthyophonus viable units (mean Â± SD) present in C-MEM cultures after 7 (A) and 14 (B) days of treatment in KZ or E and L formulations. Different letters indicate statistically

significant differences (Pâ/¤0.05) after the one-way ANOVA tests and the multiple comparisons of means. Solid lines indicate the control (C-MEM) mean values of the triplicates and dotted lines their

associated standard deviations. 209x296mm (144 x 144 DPI)


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Fig. 19. Number of Ichthyophonus colony forming units (CFU) (mean Â± SD) germinated in EFSA after previous treatment with the KZ or E and L formulations during 7 (A) or 14 (B) days. Different letters indicate statistically significant differences (Pâ/¤0.05) after the one-way ANOVA tests and the multiple comparisons of means. Solid lines indicate the control (C-MEM) mean values of the

triplicates and dotted lines their associated standard deviations. 209x296mm (144 x 144 DPI)


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Table 1. Concentrations of KZ and the other components in each dose of liposome and lipid

emulsion formulations used to evaluate Ichthyophonus growth in MEM-10 in the microplate

wells. KZ: ketoconozole. EPC: egg yolk phosphatidylcholine; CHO: cholesterol; TON:

triacylglyceride triolein.

In liposomes In lipid emulsions KZ

µg/ml Formulations EPC

mg/ml

CHO

mg/ml

Stearylamine

mg/ml

Formulations TON

mg/ml

Tween 80

mg/ml

5 KZ5L 0.014 0.005 0.001 KZ5E 0.076 0.045

50 KZ50L 0.140 0.048 0.012 KZ50E 0.757 0.447

100 KZ100L 0.280 0.095 0.024 KZ100E 1.516 0.894

200 KZ200L 0.560 0.188 0.050 KZ200E 3.040 1.766

400 KZ400L 1.120 0.376 0.100 KZ400E 6.081 3.543

0 C100L 0.380 0.095 0.024 C100E 1.607 0.894

0 C400L 1.520 0.376 0.100 C400E 6.446 3.543


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